It is well known that most of the bodily states in mammals, including most disease states, are effected by proteins. Such proteins, either acting directly or through their enzymatic functions, contribute in major proportion to many diseases in animals and man. Classical therapeutic methods have generally focused on interactions with such proteins in efforts to moderate their disease-causing or disease-potentiating functions. Recently, however, attempts have been made to moderate the actual production of such proteins by interactions with molecules that direct their synthesis, such as intracellular RNA. By interfering with the production of proteins, it has been hoped to effect therapeutic results with maximal desired effect and minimal side effects. It is the general object of such therapeutic approaches to interfere with, or otherwise modulate, gene expression leading to undesired protein formation.
One method for inhibiting specific gene expression is the use of oligonucleotides and oligonucleotide analogs as “antisense” agents. The oligonucleotides or oligonucleotide analogs complimentary to a specific, target, messenger RNA (mRNA) sequence are used. Antisense methodology is often directed to the complementary hybridization of relatively short oligonucleotides and oligonucleotide analogs to single-stranded mRNA or single-stranded DNA such that the normal, essential functions of these intracellular nucleic acids are disrupted. Hybridization is the sequence specific hydrogen bonding of oligonucleotides or oligonucleotide analogs to Watson-Crick base pairs of RNA or single-stranded DNA. Such base pairs are said to be complementary to one another.
Oligonucleotides and oligonucleotide analogs are now accepted as therapeutic agents holding great promise for therapeutic and diagnostic methods. Application of oligonucleotides and oligonucleotide analogs as antisense agents for therapeutic purposes and diagnostic purposes, and as research reagents, often requires that the oligonucleotides or oligonucleotide analogs be synthesized in large quantities.
Three principal methods have been used for the synthesis of oligonucleotides. The phosphotriester method, as described by Reese, Tetrahedron 1978, 34, 3143; the phosphoramidite method, as described by Beaucage, in Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs; Agrawal, ed.; Humana Press: Totowa, 1993, Vol. 20, 33–61; and the H-phosphonate method, as described by Froehler in Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs Agrawal, ed.; Humana Press: Totowa, 1993, Vol. 20, 63–80.
The phosphotriester approach has been widely used for solution phase synthesis, whereas the phosphoramidite and H-phosphonates strategies have found application mainly in solid phase syntheses. Reese has also reported an approach to the solution phase synthesis of oligonucleotides on H-phosphonate coupling. See, Reese et al. Nucleic Acids Research, 1999, 27, 963–971, and Reese et al. Biorg. Med. Chem. Lett. 1997, 7, 2787–2792.
In view of the growing promise of therapeutic, analytical, genomic and other uses of oligonucleotides, it is desirable to produce oligonucleotides in ever increasing quantities. A prerequisite to scaling up phosphoramidite oligonucleotide synthesis is the scale up the process of making phosphorodiamidite precursors. As impurities in the phosphorodiamidite will impact product purity, the phosphorodiamidite must be of exceptional purity. However, the classical methods of purifying phosphorodiamidite, involving distillation at elevated temperature under high vacuum (e.g. 100 degc and 0.5 mm Hg), are not economical for scale-up, as the amount of time required for distillation increases with increasing scale, which in turn leads to degradation of the phosphorodiamidite product.
There is thus a need for a scalable, economical process for purifying phosphorodiamidite.